Obesity and type 2 diabetes mellitus are two closely connected metabolic diseases that are increasing in prevalence at epidemic rates. The PI and others have shown that human obesity is associated with abnormal accumulation of lipids within the skeletal muscle cell, a phenomenon that occurs in concert with impaired insulin signal transduction. The PI and colleagues recently demonstrated that SCD1 in skeletal muscle is a core mechanism contributing to reduced fatty acid (FA) oxidation and increased intramyocellular triacylglycerol (IMTG) synthesis with obesity. To date, the transcriptional pathway(s) mediating elevated SCD1 activity in skeletal muscle of obese humans have not been elucidated. Growing evidence suggests that obesity and metabolic disorders, including insulin resistance and T2DM, are tightly associated with inflammation. Toll-like receptors (TLR) are transmembrane receptors that, upon activation, play an important role in the induction of inflammatory responses by transcriptionally activating nuclear factor kappa beta (NF-kB), a transcription factor that regulates the expression of many pro-inflammatory genes. Toll-like receptors and NF-kB have been linked to lipid-induced skeletal muscle insulin resistance. Preliminary evidence provided by the PI suggests that toll- like receptor 4 (TLR4) signaling through NF-kB modulates SCD1 transcription and lipid accumulation in skeletal muscle. SPECIFIC AIM 1: Demonstrate that TLR4 signaling through NF-kB modulates SCD1 transcription and lipid accumulation in cultures of mouse and human cell lines. SPECIFIC AIM 2: Demonstrate that TLR4 signaling through NF-kB contributes to transcriptional regulation of SCD1, lipid accumulation, and the development of insulin resistance in skeletal muscle of mice in situations of hyperlidemia. SPECIFIC AIM 3: Demonstrate that TLR4 signaling through NF-kB increases SCD1 activity and contributes to free fatty acid- induced skeletal muscle lipid accumulation and insulin resistance in humans. Specific Aim1 proposes the use of gain or loss of function strategies in cell culture to demonstrate that TLR4 and NF-kB are transcriptionally regulating SCD1. Specific Aim 2 proposes the use of TLR4 mutant (C3H/HeJ) and NF-kB knockout (nfkb1- p105) animals to demonstrate that both TLR4 and NF-kB are critically important for SCD1 regulation. Specific Aim 3 proposes to examine the role of TLR4 and NF-kB in hyperlidemic-induced skeletal muscle lipid accumulation and insulin resistance in humans. Project Narrative (Public Health Relevance): The primary objective of the current proposal is to discern the role of toll-like recpeptor-4 and nuclear factor kappa beta activation in the transcriptional regulation of stearoyl-CoA desaturase-1. Stearoyl-CoA deaturase-1 is a lipogenic enzyme that is known to contribute to lipid accumulation in skeletal muscle of obese humans. Lipid accumulation in skeletal muscle contributes to the development of insulin resistance, which is a risk factor for the development of type 2 diabetes. Importantly, the experiments proposed in the current application may lead to novel pharmacological targets for the treatment of obesity-associated insulin resistance.